Biological remediation of creosote- and similarly-contaminated sites using Pseudomonas paucimobilis

ABSTRACT

This invention concerns a biological process for remediating creosote-contaminated sites or environment sites containing polycyclic aromatic hydrocarbons generally found in creosote-contaminated sites. The biological process comprises novel bacterium, Pseudomonas paucimobilis strain EPA 505 sc., which can degrade recalcitrant chemical compounds.

This is a division, of application Ser. No. 07/371,241, filed Jun. 21,1989 just now patented, U.S. Pat. No. 5,132,224.

BACKGROUND OF THE INVENTION

In 1980, the U.S. Environmental Protection Agency concluded that wastewater from creosote and PCP wood-preserving processes poses an immediateor potential hazard to human health and the environment when improperlytreated, stored, or disposed of. Moreover, pond sediments and sludgescontaminated with wood preservatives were considered hazardous. Suchmaterials are categorized as K001 hazardous wastes (49 CFR ch.1 subpart172.101).

Creosote contamination is generally associated with surface soils,waters in treatment lagoons or evaporation areas, and groundwatercontaminated with leachate from the above sources. There areapproximately 550 sources of such waste in the United States where woodpreserving is currently conducted (Micklewright, J. T. [1986] Contractreport to the American Wood-Preserver's Institute. InternationalStatistics Council, Inc., Washington, D.C.). Collectively, activetreatment facilities generate an estimated 840 to 1530 dry metric tonsof K001 sludge annually (Sikora, L. J. [1983] In Land Treatment ofHazardous Wastes, Parr, J. G., P. B. Marsh, eds.; Noyes Data Corp., ParkRidge, N.J., pp. 397-410). Although the number of operatingwood-preserving facilities has been reduced, it has been estimated thatthere are 700 sites throughout the United States where wood preservationis, or has been, conducted (Burton, M. B., M. M. Martinson, K. D. Barr[1988] Biotech USA. 5th Ann. Indust. Conf., Nov. 14-16, San Francisco,CA). Since creosote treatment sites are commonly impacted by leakingtanks, drippings from treated lumber, spills, and leachate from unlinedholding ponds, this number presumably describes the number ofcreosote-contaminated sites as well.

A major concern when discussing creosote contamination focuses onpersistence of toxic constituents. Under appropriate conditions, allcreosote constituents are potentially degradable. Therefore, persistencetends to be a function of impregnation within the wood as opposed to aninherent recalcitrance. For example, Petrowicz and Becker (Petrowicz, H.J. and G. Beckare [1964] Materialprufung 6:461-570) demonstrated thatcreosote constituents were recovered from creosote-treated woodenblocks; 16 of these compounds were identified as naphthalene,2-methylnaphthalene, biphenyl, dimethylnaphthalene, acenaphthene,dibenzofuran, fluorene, methylfluorene, (anthracene and phenanthrene),carbazole, methylphenanthrene, 2-phenylnaphthalene, fluoranthene,pyrene, 2,3-benzo[b]fluorene, and chrysene. The same chemicals wererecovered from unweathered blocks.

Becker and Petrowicz (Becker, G. and H. J. Petrowicz [1965]Materialprufung 7:325-330) showed that more than 30 years after initialapplication, creosote-treated railroad ties exhibited only a minorchange in creosote composition. Rotard and Mailahn (Rotard, W. and W.Mailahn [1987] Anal. Chem. 59:65-69) employed more refined analyticaltechniques and found a significant amount of creosote present indiscarded railroad crossties that had been installed in playgrounds. Themost common constituents identified were (in order of decreasingconcentration) phenanthrene, anthracene, fluoranthene, pyrene, chrysene,benzo[a]pyrene, benzo[b]fluoranthene, and benzo[j]fluoranthene.

Coal-tar creosote has been widely used as a wood preservative for over150 years with an anual consumption in 1986 estimated at 454,000 metrictons (Mattraw, H. C. Jr., and B. J. Franks [1986] Chapter A."Description of hazardous waste research at a creosote works, Pensacola,FL," pp. 1-8. In A. C. Mattraw, Jr., and B. J. Franks [eds.], USGSsurvey of toxic wastes-groundwater contamination program. USGS WaterSupply Paper No. 2285). Though creosote-treated products themselves donot appear to represent a threat to the environment, accidental spillageand improper disposal of creosote at production plants and atwood-preserving facilities have resulted in extensive contamination ofsoil, surface water, and groundwater aquifers (Fisher, C. W., and G. R.Tallon [1971] Proceed. Am. Wood-Preservers' Assoc. 67:92-96; Goerlitz,D. F., D. E. Troutman, E. M. Godsy, and B. J. Franks [1986] Chapter G,pp. 49-53. USGS Water Supply Paper No. 2285). Since creosote containsmany toxic compounds and priority pollutants, such sites are consideredhazardous; hence, remedial action is required.

Recent studies have suggested that biodegradation may represent a cleanand efficient means of remediating such sites. It has also been reportedthat 85% of creosote consists of polycyclic hydrocarbons (PAH's).Therefore, biodegradation of these constituents would result in theremoval of a significant volume of creosote pollutants. Moreover, thedestruction of these components would significantly reduce the potentialhealth hazards associated wtih creosote-contaminated environments.Likewise, other environments similarly affected by PAH's (i.e., oilrefineries, coal gasification sites) may also be improved significantlyby removing the hazards associated with this class of chemicalpollutant.

Microorganisms capable of degrading certain creosote PAH's have beendescribed, and mechanisms for PAH biodegradation have been reviewed(Cerniglia, C. E., and S. K. Yang [1984] Appl. Environ. Microbiol.47:119-124). Microbial degradation of lower molecular weight PAH's suchas naphthalene and biphenyl by a variety of bacterial strains is wellestablished. Biodegradation of tricyclic compounds such as anthraceneand phenanthrene has also been reported.

There appear to be no accounts of the microbial utilization of PAH'scontaining four or more aromatic rings. However, several publicationshave described the co-metabolism of such PAH's includingbenzo[a]anthracene, benzo[a]pyrene, fluoranthene, and pyrene. Incidentalmetabolism of various PAH's by the ligninolytic fungus Phanerochaetechrysosporium grown under defined conditions has also been reported.

The basic principle of bioremediation is to exploit the ability ofmicroorganisms to catabolize a wide range of organic substrates.Trickling filtration, land-farming, activated sludge, oxidation lagoons,and soil inoculation represent a few means in which microorganisms areutilized to treat industrial wastes in situ. For bioreactor operations,engineering designs are based on the unique demands of a particularmicrobial consortium or pure culture so as to provide the idealenvironment, thereby optimizing the process. When successful,bioremediation results in the conversion of a toxic chemical tonon-toxic materials.

Though there has been a large amount of research concerning theremediation of creosote-contaminated sites, there remains a need formore effective biological systems to accomplish this goal. Theinvention, described and claimed herein, is directed to the use of novelmicrobes which can be used to remediate creosote-contaminated sites.

BRIEF SUMMARY OF THE INVENTION

The subject invention relates to the use of novel microbes to remediatecreosote- or similarly-contaminated sites. Specifically exemplified isthe use of a novel 7-membered bacterial consortium to remediatecreosote-contaminated sites. This 7-membered bacterial consortium wasisolated from a sandy soil highly contaminated with coal-tar creosote.Though isolation was accomplished by the use of an enrichment cultureemploying serial transfer through a mineral salts medium containingfluoranthene, other recalcitrant chemical compounds, for example, asdisclosed herein, can be used.

The ability of this consortium to degrade fluoranthene and otherpolycyclic aromatic hydrocarbons (PAH's) was verified by demonstratingtheir disappearance from an artificial PAH mixture using capillary gaschromatography. When grown on fluoranthene as sole carbon source andsubsequently exposed to fluoranthene plus 16 additional PAH's typical ofthose found in creosote, this consortium exhibited the capacity toremove all PAH's present in this defined mixture. After 3 days ofincubation, 13 of the original 17 PAH components were degraded to levelsbelow the limit of detection (10 ng/L). Continued incubation resulted inextensive degradation of the remaining 4 compounds. Since thisconsortium is able to utilize a high molecular weight PAH as sole carbonsource, in conjunction with its ability to transform a diverse array ofPAH's, it can be used to remediate environments contaminated with PAH'ssuch as those impacted by creosote.

We have isolated a particularly effective PAH-degrading microbe fromthis 7-membered consortium. This novel microbe has been designatedPseudomonas paucimobilis strain EPA505sc.

The microbes of the subject invention can be used in various knownprocedures for cleaning up creosote- or similarly-contaminated sites.For example, procedures such as soil percolation, activated sludge, andbioreactors can be used alone or in combination.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents gas chromatograms of methylene chloride extracts ofMS+PAH broths either un-inoculated (FIG. 1a), inoculated withPseudomonas putida [NAH7] strain PpG7 (ATCC 17485) (FIG. 1b), orinoculated with the fluoranthene-induced consortium (FIG. 1c). With theexception of two formalin-associated peaks at 3.6 and 6.5 min, the gaschromatograms of the killed-cell controls were essentially identical tothat presented in FIG. 1a. After 8 days incubation, there were nodetectable losses of PAH's from the uninoculated controls. In thepresence of PpG7, only naphthalene (peak 1) and 2-MN (peak 2) weredegraded beyond the limit of detection. The fluoranthene-inducedconsortium, however, exhibited extensive degradation of all the PAH'spresent in the defined mixture. Only fluoranthene (peak 13) and pyrene(peak 14) were present in detectable amounts (Table 3). Though thefluoranthene peak does not appear to have been significantly reduced,the area associated with this peak corresponds to 41.6% recovery which,in turn, corresponds to degradation of 0.25 mg fluoranthene in 8 days.

DETAILED DISCLOSURE OF THE INVENTION

Creosote- or similarly-contaminated sites can be remediated by use ofthe 7-membered bacterial consortium of the invention. Particularlyuseful is the isolate designated Pseudomonas paucimobilis strainEPA505sc.

A subculture of has been deposited in the permanent collection of theNorthern Research Laboratory, U.S. Department of Agriculture, Peoria,Ill., USA on Jun. 9, 1989. The accession number is as follows:

Pseudomonas paucimobilis strain EPA505sc-NRRL B-18512

The taxonomy of Pseudomonas paucimobilis strain EPA505sc is as follows:Gram-negative, aerobic, non-glucose fermenting, motile (weakly) rod(0.5×1.5 μm). Forms a 1.0 to 2.0 mm bright yellow colony on nutrientagar plus 0.5% glucose after 5 days at 28° C. Yellow pignment isnon-diffusible and non-fluorescent. Oxidizes glucose, D-gluconate, andlactose. Hydrolyses esculin. Does not assimilate arabinose, maltose,mannose, malate, N-acetyl-D-glucosamine, caprate, adipate, TWEEN™80,citrate, or phenylacetate. Does not reduce nitrate of nitrite. Negativereactions for urease, arginine dihydrolase, gelatinase, andtryptophanase.

The subject culture has been deposited under conditions that assure thataccess to the culture will be available during the pendency of thispatent application to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122. Thedeposit is available as required by foreign patent laws in countrieswherein counterparts of the subject application, or its progeny, arefiled. However, it should be understood that the availability of adeposit does not constitute a license to practice the subject inventionin derogation of patent rights granted by governmental action.

Further, the subject culture deposit will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., it will be stored with all the carenecessary to keep it viable and uncontaminated for a period of at leastfive years after the most recent request for the furnishing of a sampleof the deposit, and in any case, for a period of at least 30 (thirty)years after the date of deposit or for the enforceable life of anypatent which may issue disclosing the culture. The depositoracknowledges the duty to replace the deposit should the depository beunable to furnish a sample when requested, due to the condition of thedeposit. All restrictions on the availability to the public of thesubject culture deposit will be irrevocably removed upon the granting ofa patent disclosing it.

The novel microbes of the invention can be used, advantageously, incombination with a solubilizing agent. Examples of solubilizing agentswhich can be used in the subject invention are the many well-known andcommercially available non-ionic and anionic surface active agents anddetergents. Some examples are TWEEN™80 (a non-ionic surfactant availablefrom Fisher Chemical Co.), Merpol (a non-ionic ethylene oxide condensateproduced by E. I. duPont de Nemours and Co., Inc.), Consowet (adioctylsulfosuccinate anionic detergent produced by Consos, Inc.,Charlotte, N.C.), and Astrowet (a dioctylsulfosuccinate anionicdetergent produced by Astro American Chemical Co., Greenville, S.C.).Generally, the basic chemical structure or nature of these solubilizingagents is not limiting so long as they can be considered to be nonionicor anionic surface active agents or detergents.

The creosote-or similarly-contaminiated site degradation procedureitself, using the novel microbes isolated by the process of the subjectinvention, can be carried out by use of various known procedures. Forexample, the degradation process can be carried out by adding liquidculture media of a novel microbe to contaminated soil or water wastes.Generally, procedures as disclosed in U.S. Pat. Nos. 4,477,570 and4,483,923 can be used. As any person skilled in this art knows, goodgrowth conditions for the degrading microbes must be employed in orderto enable the microbes to degrade contaminated sites effectively.Determination of such optimum growth conditions are routine for theskilled artisan.

Following are examples which illustrate procedures, including the bestmode, for practicing the invention. These examples should not beconstrued as limiting. All percentages are by weight and all solventmixture proportions are by volume unless otherwise noted.

EXAMPLE 1 Source of Creosote-Degrading Microorganisms

Soil highly contaminated with coal-tar creosote was freshly obtainedfrom a nearby creosote-waste site in Pensacola, Fla. At one location atthis site a former evaporation pond for creosote-contaminated wastewater resulted in the formation of a 2-inch layer or tar-like sludgeheavily contaminated with more than 50% (weight) methylenechloride-extractable organics. This layer was located approximately 6inches below the soil surface. Soil immediately adjacent to this sludgewas collected from depths of 4 to 8 inches and was used as the source ofmicroorganisms. Detailed reports on the history of creosote use, typeand amount of pollutants present, and the extent of environmentalcontamination at this site are available (Godsy, E. M., and D. F.Goerlitz [1986] In A. C. Mattraw, Jr., and B. J. Franks [eds.], USGSsurvey of toxic wastes--groundwater contamination program. USGS WaterSupply Paper No. 2285, Chapter H, pp. 55-58; Pereira, W. E., and C. E.Rostad [1986] USGS Water Supply Paper No. 2285, Chapter E, pp. 33-40;Troutman, D. E., E. M. Godsy, D. F. Goerlitz, and G. G. Ehrlich [1984]"Phenolic contamination in the sand and gravel aquifer from a surfaceimpoundment of wood treatment wastes, Pensacola, Fla., " USGS WaterResources Invest. Report No. 84-4231, 36p.)

EXAMPLE 2 Mineral Salts+PAH Medium

The mineral salts (MS) medium used consisted of (mg/L): (NH₄)₂ SO₄=1000; KH₂ PO₄ =200; MgSO₄.7H₂ O =200; CaCl₂.2H₂ O=100; FeCl₃.6H₂ O=5;(NH₄)₆ Mo₄ O₂₄.4H₂ O=1. To achieve the aqueous PAH concentrationsreported in Table 1, TWEEN™80 (Fisher Chemical Co.) was added at 200mg/L. The pH was adjusted to pH=7.0 with 0.1N HCl and the medium wassterilized (104 kPa, 121° C., 20 min.) prior to the addition of organicsubstrates. Polycyclic aromatic hydrocarbons (Sigma Chemical Company)used were of the highest purity (>98%) available).

To prepare an aqueous solution containing a defined mixture of PAH'sclosely related to the PAH composition of creosote, the appropriateamount of each compound (Table 1) was added to a sterile flask anddissolved in 5.0 ml methylene chloride to effect sterilization.Methylene chloride was removed under a stream of dry nitrogen passedthrough a 0.25 μm filter and the PAH's were dissolved by mixing with amagnetic stir bar into an appropriate amount of sterile MS medium. Aftermixing for 6 hours at room temperature, the medium designated MS+PAH wasfiltered through a layer of sterile glass wool to remove undissolvedsolids. Medium was stored at 1° C. in sterile, 1.0 L Wheaton bottlesfitted with Teflon-lined screw-caps. The concentration of each compoundwas determined by capillary gas chromatography of extracted samples asdescribed in following sections.

                                      TABLE 1                                     __________________________________________________________________________    Composition of a defined polycyclic aromatic hydrocarbon (PAH) mixture        and its relationship to predominant PAH's found in coal-tar creosote.                                       PAH concentration in                                                    Aqueous      coal tar                                                         solubility.sup.2                                                                    defined PAH                                                                          creosote.sup.4                           Peak             abbreviation                                                                         (25° C.)                                                                     mixture.sup.3                                                                        range % -                                number.sup.1                                                                       compound    (if used)                                                                            mg/L  mg/L   total PAH                                __________________________________________________________________________    1    naphthalene --     31.7  17.1    3.0-15.8                                2    2-methylnaphthalene                                                                       2-MN   25.4  17.1    2.1-14.2                                3    1-methylnaphthalene                                                                       1-MN   28.5  16.0    2.1-14.2                                4    biphenyl    --     7.5   5.8    2.3-2.8                                  5    2,6-dimethylnaphthalene                                                                   2,6-DMN                                                                              2.0   2.1    2.0-2.3                                  6    2,3-dimethylnaphthalene                                                                   2,3-DMN                                                                              3.0   1.9    2.0-2.4                                  7    acenaphthene                                                                              --     3.9   3.8    4.1-9.0                                  8    fluorene    --     2.0   3.9     8.6-10.0                                9    phenanthrene                                                                              --     1.3   7.0     4.6-21.0                                10   anthracene  --     0.07  2.7    1.5-2.0                                  11   2-methylanthracene                                                                        2-MA   0.04  0.2    0.5-2.6                                  12   anthraquinone                                                                             --     --    0.9    0.1-1.0                                  13   fluoranthene                                                                              --     0.26  8.7     6.8-10.4                                14   pyrene      --     0.14  2.3    2.2-8.5                                  15   2,3-benzo[b]fluorene                                                                      2,3-BBF                                                                              0.002 0.4    2.0-4.6                                  16   chrysene    --     0.002 0.3    2.8-3.0                                  17   benzo[a]pyrene                                                                            BAP    0.003 1.2    0.1-1.0                                       TOTAL:                   91.4                                            __________________________________________________________________________     .sup.1 order of elution through capillary column SBP5 (Supelco)               .sup.2 Baker, R. J., W. E. Acree, Jr., and C. C. Tsai (1984) Quant.           Struct. Act. Relat. 3:10-16; Mackay, D., and W. Y. Shiu (1977) J. Chem.       Eng. Data 22:399-402.                                                         .sup.3 Increased solubility in the presence of 200 mg/L TWEEN ™ 80         .sup.4 Ranges based on analyses by Andersson, K., J. O. Levin, and C. A.      Nilsson (1983) Chemosphere 12:197-207; Becker, G. (1977) Proceed. Am.         WoodPreservers' Assoc. 73:16-25; Borowitzky, H., and G. Schomburg (1979)      J. Chrom. 170:99-124; Lorenz, L. J., and L. R. Gjovik (1972) Proceed. Am.     WoodPreservers' Assoc. 68:32-41; Nestler, F. H. M. (1974) Fuel 60:213-220     Novotny, M., J. W. Strand, S. L. Smith, D. Wiesler, and F. J. Schwende        (1981) Fuel 60:213-220.                                                  

EXAMPLE 3 Fluoranthene (A Recalcitrant Chemical) Enrichment Cultures

A MS+fluoranthene medium was prepared according to the protocol inExample 1, with the following modifications: (1) an excess offluoranthene (approximately 500 mg/L) was supplemented for the otherorganic components shown in Table 1; (2) suspended solids were notremoved; and (3) TWEEN™80 was not added. Fifty ml of this medium weretransferred to a 250 ml screw-cap Erlenmeyer flask and inoculated with1.0 g (wet weight) creosote-contaminated soil passed through a 50-meshsieve. Flasks were incubated in the dark (28±1° C., 175 cycles/min)under controlled conditions. After 5 days incubation, a 5.0 ml aliquotwas diluted 1:10 (vol/vol) with fresh MS+fluoranthene broth andincubated for 3 days. Subsequent samples were diluted 1:50 with the samemedium every 3 days. Following several such transfers, disappearance ofundissolved fluoranthene crystals was visually apparent.Fluoranthene-utilizers were maintained by regularly diluting establishedcultures 1:50 with fresh MS+fluoranthene broth every 14 days.

EXAMPLE 4 Partial Characterization of Fluoranthene-Utilizing MicrobeConsortium

Numerous aliquots from fluoranthene-enrichment cultures of various ageswere streaked for isolation on Nutrient Agar (Difco, Detroit, Mich.)amended with 0.5% glucose (NAG agar). After 5-14 days incubation at 28°C., colonies representative of each of the different morphological typeswere removed and the single colonies repeatedly purified on NAG agar.Ultimately, 7 morphologically distinct, Gram-negative bacteria wereisolated in pure culture. These organisms were designated EPA50FAE 1, 2,3, 4, 5, 5b, and 6.

To ensure that all organisms essential for fluoranthene-utilization hadbeen isolated, MS+fluoranthene broth was inoculated with all sevenisolates to reconstitute the consortium, and fluoranthene degradationwas assessed. The consortium was reconstituted by removing singlecolonies is each organism from NAG plates and suspending them in sterileMS medium to uniform density (%T₆₀₀ =50±2.0). Fifty ml ofMS+fluoranthene broth were inoculated with 0.2 ml of each suspension andincubated at 28° C. with aeration (175 cycles/min). Fluorantheneutilization was qualitatively assessed by recording visually apparentincreases in microbial biomass, spectral (color) changes, anddisappearance of fluoranthene crystals. The sequence of color changes inthe medium was from colorless to bright orange to bright yellow to alight brown which was maintained after fluoranthene crystals were nolonger visible. Exhausted cultures to which additional fluoranthene wasadded completed this sequence in two days. Qualitative increases inmicrobial biomass were evident. However, since the fluoranthene culturesexhibited a strong tendency to form rapidly settling clumps, increasesin microbial biomass could not be measured quantitatively.

When plated on a complex medium such as NAG agar (Nutrient Agar, Difco,amended with 0.5% glucose), a total of seven morphologically distinct,Gram-negative bacteria were isolated. This 7-membered consortiummaintained its integrity throughout the enrichment procedure and throughrepeated serial transfers, thereby reflecting stability. WhenMS+fluoranthene broth was inoculated with the reconstituted consortium,fluoranthene degradation was again obvious. However, there was aninitial lag of 5 to 7 days before fluoranthene degradation becameapparent. After this period, fluoranthene degradation was rapid (<2days).

Table 2 summarizes percent recovery from MS+PAH broths of 17 PAH'spresent in the defined mixture 3 days after inoculation with either thefluoranthene-induced consortium or with P. putida PpG7. Extractionefficiencies and losses attributable to abiotic processes were accountedfor by comparing recovery values for each compound with that obtainedfrom the killed cell controls. With the exception of naphthalene (84.3%)and 2,3-DMN (78.9%), percent recovery from abiotic controls was greaterthan 85%.

The ability to detect selective utilization of individual components ofthe defined mixture was demonstrated with the culture inoculated withPpG7. After 3 days incubation, only naphthalene and 2-MN wereextensively degraded. These data were identical to those obtained after5, 8, and 14 days incubation (data not shown).

When the consortium was grown on fluoranthene and subsequently exposedto fluoranthene plus 16 other PAH's, the fluoranthene-induced consortiumexhibited the ability to degrade all of the PAH's present in the definedmixture (Table 2). After 3 days incubation, 13 of the original 17 PAH'swere degraded below the limits of detection (10 ng/L). Additionally,greater than 90% degradation of anthracene and anthraquinone wasevidenced by their percent recoveries, 1.5 (±1.5) and 6.5 (±6.5),respectively. The remaining 2 compounds, fluoranthene and pyrene, werealso degraded as demonstrated by respective recoveries of 69.5 (±13.6)and 44.3 (±8.5).

                  TABLE 2                                                         ______________________________________                                        Biodegradation of 17 PAH's by a 7-membered, fluoranthene-                     induced bacterial consortium isolated from a creosote waste site.                     % recovery of select PAH's from broth                                         culture after 3 day incubation with                                             Fluoranthene-                                                                 induced    Killed cell                                              Compound.sup.1                                                                          consortium control.sup.2                                                                              P. putida PpG7                              ______________________________________                                        naphthalene                                                                             .sup. ND.sup.3                                                                            84.3 (± 2.3)                                                                          ND                                           2-MN      ND          85.7 (± 10.3)                                                                         ND                                           1-MN      ND          85.2 (± 7.3)                                                                           64.5 (± 3.0)                             biphenyl  ND          86.6 (± 13.8)                                                                          89.7 (± 1.8)                             2,6-DMN   ND          91.4 (± 4.8)                                                                           97.1 (± 2.9)                             2,3-DMN   ND          78.9 (± 12.7)                                                                          82.5 (± 2.5)                             acenaphthene                                                                            ND          96.3 (± 3.7)                                                                          102.4 (± 0.8)                             fluorene  ND         106.2 (± 11.8)                                                                         117.8 (± 10.7)                            phenanthrene                                                                            ND          91.2 (± 10.8)                                                                         119.4 (± 11.4)                            anthracene                                                                              1.5 (± 1.5)                                                                            91.5 (± 4.1)                                                                          100.6 (± 1.6)                             2-MA      ND         100.0 (± 30.0)                                                                          67.0 (± 2.0)                             anthraquinone                                                                           6.5 (± 6.5)                                                                            88.9 (±  85.6 (± 14.8)                            fluoranthene                                                                            69.5 (± 13.6)                                                                         101.2 (± 9.8)                                                                          102.1 (± 18.8)                            pyrene    44.3 (± 8.6)                                                                           87.0 (± 8.7)                                                                          108.9 (± 8.1)                             23-BBF    ND         115.0 (± 15.0)                                                                         120.0 (± 5.0)                             chrysene  ND         106.7 (± 16.3)                                                                         126.7 (± 7.7)                             benzo[a]pyrene                                                                          ND         115.6 (± 7.7)                                                                          114.0 (± 14.2)                            ______________________________________                                         .sup.1 See Table 1 for abbreviations used.                                    .sup.2 fluorantheneinduced bacterial consortium killed with 5%                formaldehyde (37% formalin solution) at the time of inoculation.              .sup.3 ND = not detected (<0.01 mg/L).                                   

With continued incubation, further degradation of the 4 compounds whichwere still present at 3 days was observed (Table 3). Following 5 days ofincubation, anthracene and anthraquinone were no longer recoverable. Theamount of fluoranthene extractable after 5, 8, and 14 days incubationdecreased from 52.1 to 41.6 to 16.8%, respectively. Similarly, recoveryof pyrene after 5, 8, and 14 days incubation decreased from 43.9 to 17.4to 12.0%, respectively.

The relatively high recovery of fluoranthene from tubes inoculated withthe fluoranthene-induced control requires clarification. Consortiumbiomass for inoculation was generated in MS+fluoranthene broth whichcontained an excess of insoluble fluoranthene (500 mg/L). It was laterdetermined that there was a significant carry-over of fluoranthene fromthe cultures. It could be calculated that those tubes inoculated withthe fluoranthene consortium received an additional 0.38 mg offluoranthene (75.1 mg/L) resulting in an initial fluorantheneconcentration of 83.8 mg/L. Therefore, after 3 days incubation, thefluorantheneinduced consortium had degraded 30% of the total amount offluoranthene originally present or 0.13 mg fluoranthene.

                  TABLE 3                                                         ______________________________________                                        Continued loss of PAH's remaining after 3 days incubation.                            % recovery of PAH's after extended incubation                                 with the fluoranthene-induced consortium                              Compound  Day 3     Day 5     Day 8  Day 14                                   ______________________________________                                        anthracene                                                                               1.5      ND.sup.1  ND     ND                                                 (± 1.5)                                                          anthraquinone                                                                            6.5      ND.sup.   ND     ND                                                 (± 6.5)                                                          fluoranthene                                                                            69.5      52.1      41.6   16.8 (± 6)                                       (± 13.6)                                                                            (± 22.3)                                                                             (± 8.8)                                      pyrene    44.3      43.9      17.4   12.0 (± 12)                                     (± 8.6)                                                                              (± 12.6)                                                                             (± 7.0)                                      ______________________________________                                         .sup.1 ND = not detected (<0.01 mg/L).                                   

The following Table 4 gives a partial characterization of the bacterialconsortium:

                  TABLE 4                                                         ______________________________________                                        Partial characterization of the bacteria                                      comprising the fluoranthene-utilizing community.                              Strain                                                                        designation                                                                           Colony morphology.sup.1                                                                            Gram reaction                                    ______________________________________                                        FAE1    white, 1-2 mm mucoid negative rods                                    FAE2    light brown, 3-4 mm, mucoid                                                                        negative cocci                                   FAE3    colorless, 1-2 mm, mucoid                                                                          negative cocci                                   FAE4    white, 3-4 mm, slime producing                                                                     negative rods                                    FAE5    bright yellow, 1-2 mm, mucoid                                                                      negative rods                                    FAE5b   opaque yellow, <1 mm, mucoid                                                                       negative rods                                    FAE6    white, 4-5 mm, spreading                                                                           positive cocci                                   ______________________________________                                         .sup.1 Colony morphology after 5 days incubation at 28° C. on NAG      agar.                                                                    

EXAMPLE 5 Preparation of Fluoranthene-Induced Cell Suspensions ofConsortium

Fifty ml of MS+fluoranthene broth were transferred aseptically to aclean, sterile 250 ml Erlenmeyer flask fitted with Teflon-linedscrew-caps, inoculated with the reconstituted bacterial consortium, andincubated (28° C., 175 cycles/min) for 10 days. After 10 daysincubation, cultures were diluted 1:100 (vol/vol) in freshMS+fluoranthene broth. Fluoranthene degradation was visually apparentafter 2 days incubation at which time the consortium was diluted 1:25.Following 3 days incubation, fluoranthene-induced cells wereconcentrated (10,000 g, 10 min, 4° C.) and resuspended in 1/10 vol MSmedium.

EXAMPLE 6 Action of Consortium Cells Towards PAH's

Suspensions of fluoranthene-induced cells of the consortium (100 μl)were used to inoculate 5.0 ml MS+PAH broth in clean, sterile 50.0 mltest tubes fitted with Teflon-lined screw-caps. Killed cell controlswere generated by adding 250 μl of a 37% formalin solution to 8 of the16 tubes inoculated with the fluoranthene-induced consortium.Uninoculated controls were also incorporated. In addition, 8 tubescontaining MS+PAH broth were inoculated with 100 μl of cell suspensionof Pseudomonas putida PpG7 (a gift from Dr. I. C. Gunsalus, Universityof Illiniois) which, in preliminary studies, demonstrated the ability toselectively utilize only 2 compounds (naphthalene and2-methylnaphthalene) present in the defined PAH mixture. After 3, 5, 8,and 14 days incubation (28° C., 200 cycles/min), duplicate tubes of eachtreatment were removed and extracted from determination of PAH'spresent.

EXAMPLE 7 Methylene Chloride Extraction Procedure

At selected times, MS+PAH broth in a given tube was transferred to aclean, methylene chloride-rinsed, 15 ml glass conical extraction tubefitted with a Teflon-lined screw-cap. The original incubation tube wasrinsed with 2.0 ml methylene chloride which was added to the extractiontube. Tubes were shaken for 1.0 min to facilitate the extraction ofunmetabolized PAH's into the organic phase. Methylene chloride wasseparated from the aqueous phase after centrifugation (2500 g, 5 min).The entire separated organic phase (2.0 ml) was removed employing amethylene chloride-rinsed, 1.0 ml glass syringe fitted with a blunt-endneedle, and transferred to a clean, solvent-rinsed concentration tube.The extraction procedure was repeated 2 more times with 0.5 ml methylenechloride. The final volume of methylene chloride (3.0 ml) was reduced to<1.0 ml under a stream of dry nitrogen. After the final volume ofmethylene chloride was adjusted to 1.0 ml, each extract was spiked with10 μl of a 1,4-naphthaquinone solution (10 mg/ml methylene chloride) asa marker and transferred to a GC vial for subsequent analysis.

EXAMPLE 8 Capillary Gas Chromatography

Gas chromatographic analysis of methylene chloride extracts and ofindividual PAH standards was performed on a Hewlett-Packard model 5710Agas chromatographic equipped with a flame ionization detector. Hydrogenwas used as carrier gas (0.5 ml/min) while air (240 ml/min) and hydrogen(30 ml/min) was supplied for the flame ionization detector. Polycyclicaromatic hydrocarbons in replicate 1.0 μl injections were separated on a15.0 m×0.32 mm I.D. SPB-5 (Supelco, Bellefonte, Pa.) capillary columnwith a 0.25 μm coating phase. Oven temperature was programmed at 80° C.for 2 min followed by a linear increase of 8° C./min to 280° C. where itwas held for 4 min (30 min run). Injector and detector temperatures weremaintained at 270° C. Percent recovery of each PAH was calculated bycomparing peak area with that of standards for each compound.

We claim:
 1. A biologically pure culture of Pseudomonas paucimobilisstrain EPA505sc., having all of the identifying characteristics ofdeposit NRRL B-18512.